Trackoscope: A low-cost, open, autonomous tracking microscope for long-term observations of microscale organisms

Cells and microorganisms are motile, yet the stationary nature of conventional microscopes impedes comprehensive, long-term behavioral and biomechanical analysis. The limitations are twofold: a narrow focus permits high-resolution imaging but sacrifices the broader context of organism behavior, while a wider focus compromises microscopic detail. This trade-off is especially problematic when investigating rapidly motile ciliates, which often have to be confined to small volumes between coverslips affecting their natural behavior. To address this challenge, we introduce Trackoscope, a 2-axis autonomous tracking microscope designed to follow swimming organisms ranging from 10μm to 2mm across a 325cm2 area (equivalent to an A5 sheet) for extended durations—ranging from hours to days—at high resolution. Utilizing Trackoscope, we captured a diverse array of behaviors, from the air-water swimming locomotion of Amoeba to bacterial hunting dynamics in Actinosphaerium, walking gait in Tardigrada, and binary fission in motile Blepharisma. Trackoscope is a cost-effective solution well-suited for diverse settings, from high school labs to resource-constrained research environments. Its capability to capture diverse behaviors in larger, more realistic ecosystems extends our understanding of the physics of living systems. The low-cost, open architecture democratizes scientific discovery, offering a dynamic window into the lives of previously inaccessible small aquatic organisms.

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We have used figshare to upload and link to our code database.GitHub is still used for the code as it implements version control and the figshare links to it.figshare.com/projects/Trackoscope_A_Low-Cost_Open_Autonomous_Tracking_Microscope_for_Long-Term_Observations_of_Microscale_Organisms/200934and github.com/bhamla-lab/Trackoscope8. Please review your reference list to ensure that it is complete and correct.If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript.If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.
We have reviewed references.Three references (15,28,29) have been added regarding the addition of brine shrimp tracks and an additional frugal device paper.
Responses to comments from Reviewer 1: Dear Authors, the designed system is well described in the manuscript, and all the documents uploaded to GitHub are accessible in a usable format.Firstly, the range of the Trackoscope which is designed motorized X and Y stages with an 18 cm x 18 cm travel range, is excellent.Additionally, the microstepping mechanism is a very useful setup for the device.The Optical Core is designed with adequate quality, and not requiring a high-performance GPU when tracing organisms with OpenCV's Channel and Spatial Reliability Tracker (CSRT tracker) makes the design very efficient.
We would like to thank the reviewer for their feedback and appreciation for the tracking range and simpleness.
In terms of the Performance Evaluation of Tracking and Imaging Capabilities, the resolution for slowmotion organisms is good.However, the resolution for fast organisms is comparatively lower than that of other systems.Nevertheless, it's important to note that there is currently no setup available, except yours, that can effectively trace these organisms at both large spatial and time scales.
Thank you for the feedback, we acknowledge this and note that this resolution is due to the versatility and simpleness of the CSRT and the low-cost actuators.
Please correct the species name in the supplementary files to italic and small-capital letters.
Thank you for pointing this out, we have adjusted the species names accordingly.For Trackoscope's Customizable Design for Wider Use: I recommend adding an additional supplementary file to the manuscript, demonstrating the tracing of a multicellular organism like Botryllid ascidian (marine invertebrate) larvae or adult oral siphon movements (such as feeding behavior) or zooplankton movements.This addition could attract wider attention from the scientific community.
We thank the reviewer for making this suggestion.We have already included tardigrades as a multicellular organism.But we agree that adding other multicellular organisms will attract wider attention.Therefore, we have also conducted tracks with brine shrimp, a type of zooplankton, and have added a supplementary video showcasing a track of brine shrimp nauplii in darkfield.
Tracking of brine shrimp nauplii emphasizes their chaotic and random movements.In the nauplii stage, brine shrimp are still adjusting to movement with front jointed antennae and their motion is not smooth as their phyllopods (swimming appendages) have not developed yet [27].In the track, we observe circular lurching motion [28] as the nauplii moves at an average speed of 515 μm/s with a top speed of 1200 μm/s (1.2 body lengths/second) (S6 Video).As the nauplii attempts to move linearly, it tends to move in a clockwise circle suggesting that its left antenna is stronger than the right; when coordination is achieved, fast linear motion is observed.(lines 216-223, page 8) Responses to comments from Reviewer 2: This is excellent paper demonstrating the design of a cheap and simply to build tracking microscope.It seems to me that a lot of thought has been given to its design and execution.I highly recommend the publication of this manuscript.
We thank the reviewers for their kind words and appreciation for the device.
I have one comment: The final tracks look a bit rough.The tracking dot moves around on the organism a lot.This can be fixed in post-processing I suppose?I.e. the video is saved (using Open Broadcast Studio) and then subsequent analysis could fine-tune the tracking precision.It would be nice if such a process could be demonstrated, but is definite not necessary for the publication of this manuscript.(more of a nice to have, than need to have).
We acknowledge that the tracks are slightly rough.This is due to the simple and versatile nature of the OpenCV CSRT as it constantly adapts its model throughout the track from the user's initial 1 frame selection.This adaptation over the course of the track can lead to the tracking dot shifting around on the organism as observed.However, with the versatility and adaptability of the tracker, the tracking dot tends to stay on the organism, even though it is not always the center of the organism.We have added this clarification to the paper.You are correct in assuming that this can be corrected in post-processing, but this would require a separate custom software that takes in the video and position output from Trackoscope to then adjust the tracking values based on the video.

The CSRT model updates the bounding box by continually adapting over the duration of the track as it considers both the appearance of the organism and its motion characteristics. This adaptability makes the CSRT particularly effective in handling challenges such as occlusion, rotation, and scale variation. (lines 96-99, page 5)
Additionally, as the CSRT model evolves over the track, the perceived center of the organism may not always be accurate or precise; this is another factor for the incorporation of a central zone as the perceived center, while always on the organism, may not be on the exact center.(lines 106-109, page 5) Responses to comments from Reviewer 3: Priya Soneji and authors describe Trackoscope, an automated tracking microscope enabling longduration observations of motile microorganisms.The authors highlight limitations of conventional microscopes in studying behaviors of swimming microorganisms over extended time periods.They introduce a novel tracking microscope design utilizing low-cost, modular hardware components and custom (open) software implementing open-source computer vision algorithms.The system demonstrates impressive microscopic resolution down to 8.77 μm and adaptable speed tracking from 0.02 to 11.8 body lengths/sec across diverse organisms with unique behaviors.They limit their exploration to 2D, and discuss the complexity and cost associated with motorized Z stages-while showcasing the data accessible in these dimensions.Examples showcase insights into feeding, morphology changes, reproduction, and comparative locomotion.As an open, flexible platform made with economical parts, I believe Trackoscope promises to make automated microscopy more accessible for research and education.
We would like to thank the reviewer for the time and effort required to provide such detailed comments, especially on the low-cost open nature of Trackoscope, its resolution, and overall design.

Comments:
The Trackoscope design fits well into the growing ecosystem of open hardware/software instrumentation.Trackoscope provides functionality comparable to expensive commercial systems at a fraction of the cost, while utilizing open libraries such as OpenCV and others in the Python universe that enable customization without reliance on proprietary software.The integration of hardware and software components to quickly enable versatile automated tracking of cells will be useful to many researchers and educators.
The resolution analysis confirms capabilities for resolving microorganism cells and structures.Speed profiling indicates suitable performance across crawling, swimming, and ciliates with rapidly changing behavior.
Diverse organism tracking experiments highlight new behavioral insights uniquely enabled by Trackoscope's combination of resolution, field of view, and duration.
An accessible and customizable platform promotes adoption by researchers with limited budgets.Open design fosters educational uses and customization.The discussion of multiple low-cost materials such as MDF makes the project adaptable to many communities.
We thank the reviewer for their appreciation of the modularity of Trackoscope, and its tracking versatility.

Minor Comments:
Additional details on tracking accuracy, effect of illumination, and computational analysis methods would further strengthen characterizations of organism behavior.As the system seems catered to ciliates, the integration of a simple dark field illumination could be uniquely beneficial for these studies with Trackoscope.This could significantly improve visualization of low-contrast transparency features in protists, like pellicles, membranes, and cytoskeletal elements.This is a minor comment however, as the system is designed with frugality in mind, and the simplicity of the illumination setup is understandablethe open and customizable optics design of Trackoscope allows flexibility in illumination methods, future iterations could investigate more imaging modes.
We appreciate the reviewer's feedback.We agree that additional illumination modes would allow for better visibility.We have added data with dark field illumination for Spirostomum and Brine Shrimp in supplementary videos 2, 3, and 5 and have modified Figure 2 to include an example of darkfield illumination to visibly show the effect of illumination.
Dark field illumination can also be used with Trackoscope by adding a 3D printed attachment to the ring light that directs the light around the sample from below.(lines 80-82, pages 4-5) Dark field imaging allows for higher contrast when observing more translucent organisms (Fig 2c and S2 Video).(lines 128-129, page 6) Regarding the comment about tracking accuracy, we found that such a metric is hard to quantify.The OpenCV CSRT constantly adapts its model over the duration of the track leading to some error.So while the perceived center will still be on the organism's body, it may not always be the precise center.We have added this clarification to the paper as well as more detail on the adaptability of the tracker.
Additionally, as the CSRT model evolves over the track, the perceived center of the organism may not always be accurate or precise; this is another factor for the incorporation of a central zone as the perceived center, while always on the organism, may not be on the exact center.(lines 106-109, page 5) Figures are clear and simple, I could not find anything that requires major improvement-besides the request that the text in the plot legends be increased in size (as much as possible) for clarity in reading.The plot ticks in the amoeba data in figure 5 are especially small.We thank the reviewer for this feedback and have increased the font size in Figure 5.
Overall Evaluation: I recommend this manuscript for publication.The authors have designed, validated, and demonstrated a novel tracking microscope that overcomes limitations of conventional designs.Unique insights into microorganism locomotion and behaviors are enabled.The open, flexible architecture promises to expand access to automated microscopy.The integration of the many powerful python-based computer vision libraries, while discussing the benefits of simplified methods such as CSRT, are appreciated.While machine learning techniques like convolutional neural networks have revolutionized computer vision, they also have disadvantages like requiring large training datasets, extensive computational resources, and loss of interpretability.Trackoscope displays how efficient deployment of robust open-source tools can sidestep these issues and provide reliable tracking performance without complexity.Avoiding sophisticated neural networks in favor of classical CV algorithms aligns well with the overall aims of an accessible, adaptable platform.This work represents a valuable contribution with both scientific and educational impacts in the microscopy field.
We thank the reviewer for their encouraging comments and their time.